The invention concerns an improved amphotropic retroviral packaging cell line, a process for its production as well as its use especially for gene therapy in vitro and in vivo. High virus titres can be achieved with retroviral vectors which are produced by such a packaging cell line.
Retroviral vectors are widely used to transfer foreign genes into cells of mammals and humans. However, for safety reasons such retroviral vectors should no longer be replication-competent. For this reason packaging cell lines are used to produce retroviral vectors which are not able to produce wild-type virus since they contain no functional packaging sequences. Such packaging cell lines have been known and described for a long time: xcexa82 (Mann et al., Cell 33 (1983) 153-156), xcexa8-Am (Cone and Mulligan, Proc. Natl. Acad. Sci. USA 81 (1994) 6349-6353), PA12 (Miller et al., Mol. Cell Biol. 5 (1985) 431-437), GP+E-86 (Markowitz et al., J. Virol. 62 (1988) 1120-1124), PA317 (Miller and Buttimore, Mol. Cell Biol.6 (1986) 2895-2902), and GP+env Am12 (Markowitz et al., Virology 167 (1988) 400-406), see also xe2x80x9cHandbuch der molekularen Medizinxe2x80x9d, Vol. 1 Ed. D. Ganten and K. Ruckpaul, Springer Publishers, Heidelberg 1997, R. Rxc3xcger, chapter 2.1, 197-241.
However, all these packaging cell lines except for GP+env Am12 (referred to as Am12 in the following) lead to a certain extent to the formation of wild-type virus (Bosselman et al., Mol.Cell Biol. 7 (1987) 1797-1804). In contrast Am12 in which the viral gag and pol functions are introduced into the cell on one plasmid and env is introduced into the cell on another plasmid, is a safe packaging cell line. These functions are therefore integrated in different loci of the packaging cell genome. In order to form Nvld-type virus from Am12 three recombinations have to occur which restore the original intact genome. This appears to be almost impossible and also has previously not been observed.
In order to avoid the problems caused by homologous recombination in packaging cell lines attempts have for example also been made to express gag/pol and env by different promoters (Meyers et al., Arch.Virol. 119 (1991) 257-264; Dougherty et al., J.Virol. 63 (1989) 3209-3212) and to use packaging cell lines based on spleen necrosis virus (SNV) (Mlartinez and Dornburg, Virology 208 (1995) 234-241).
However, a disadvantage of the known packaging cell lines is that the vector-virus titres of the retroviral vectors produced in this manner are very low and range between 102 and a maximum of 106 colony-forming units (CFU)/ml cell culture supernatant.
The object of the invention is to provide retroviral, safe packaging cell lines which are able to produce vector viruses with a high transduction efficiency.
The invention concerns an amphotropic retroviral packaging cell line which contains functional gag, pol and env genes integrated into the genome in which the expression of the genes gag and pol is regulated independently of the expression of the env genes characterized in that the genome of the said cell line contains one or two functionally active env genes.
A further subject matter of the invention is a process for producing a retroviral vector characterized in that an amphotropic retroviral packaging cell line according to the invention is transfected with a vector-virus (vector genome) which contains one or more genes that are heterologous for the virus but contains no functionally active retroviral structural genes in which the cell line is cultured and the retroviral vector is isolated from the culture supernatant.
A further subject matter of the invention is a process for producing an amphotropic retroviral packaging cell line characterized in that an env helper plasmid containing a selection gene and a gag/pol helper plasmid containing a selection gene are transfected into a eukaryotic cell, transfected cells are identified on the basis of the selection gene and are isolated and those cells are selected which contain one or two functionally-active env genes.
A further subject matter of the invention is a replication-incompetent retroviral vector virus which has been produced by the method according to the invention.
It has turned out that the number of env integrates in the genome of a packaging cell line has a decisive influence on the transduction efficiency of the vector-viruses produced in this manner. This is all the more surprising since it is known from Martinez and Dornburg, Virology 208 (1995) 234-241 that the extent of env expression in a packaging cell line would not have an influence on the transduction efficiency. The packaging cell lines examined by Martinez and Dornburg differ with respect to env expression only in the promoter strength of the sequences regulating env expression.
In a preferred embodiment the packaging cell line according to the invention contains two env integrates and a gag/pol integrate. According to the invention use of the packaging cell line achieves a titre of retroviral vectors of 2 106 to 107 or more, preferably at least 107 colony-forming units/ml cell culture supernatant and/or preferably a transduction efficiency of at least 50% in MOLT-4 cells (ATCC CRL 1582) after three days measured by the centrifugation method.
The independent regulation of the gene expression of gag/pol and env is usually achieved by locating at least one expression control sequence and/or a stop signal between gag/pol and env.
Starting cell lines for the packaging cell line are for example 3T3, D17. Suitable starting vectors are based on, for example, MLV, MoMuLV. Such suitable packaging cell lines and starting vectors are known to a person skilled in the art and are described for example by R. Rxc3xcger (1997) in xe2x80x9cHandbuch der molekularen Medizinxe2x80x9d.
The cell line HSR BM01 is particularly preferred as a packaging cell line according to the invention. This cell line was deposited on Dec. 9, 1995 by Boehringer Mannheim GmbH according to the Budapest Contract in the xe2x80x9cDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbHxe2x80x9d (DSM), Mascheroder Weg 1b, 38124 Braunschweig under the No. DSM ACC 2235.
A further subject matter of the invention is a process for producing an amphotropic retroviral packaging cell line according to the invention.
An amphotropic split genome retroviral packaging cell line can for example be produced as described in Markowitz et al., Virology 167 (1988) 400-406. Two plasmids are used for this which code for the so-called f helper sequences (gag, pol and env).
In order to select cell lines according to the invention, the number of env and gag/pol integrates has to be determined. This is expediently carried out by digesting the genomic DNA with restriction enzymes which do not cleave in the gag/pol sequences or env sequences and determining the number of fragments which contain gag/pol- or env-specific sequences.